1. Field of the Invention
The present invention is directed to novel cellulase compositions which share unique highly conserved regions with a known useful cellulase. More specifically, the present invention relates to a series of newly discovered enzymes from fungi and bacteria which are related by virtue of having at least one of five important conserved amino acid sequences which are also present in EGIII.
2. State of the Art
Cellulases are enzymes which are capable of hydrolysis of the β-D-glucosidic linkages in celluloses. Cellulolytic enzymes have been traditionally divided into three major classes: endoglucanases, exoglucanases or cellobiohydrolases and β-glucosidases (Knowles, J. et al., (1987), TIBTECH 5, 255-261); and are known to be produced by a large number of bacteria, yeasts and fungi.
Primary among the applications that have been developed for the use of cellulolytic enzymes are those involving degrading (wood) cellulose pulp into sugars for (bio)ethanol production, textile treatments like ‘stone washing’ and ‘biopolishing’, and in detergent compositions. Thus, cellulases are known to be useful in the treatment of mechanical pulp (see e.g., PCT Publication No. WO 92/16687). Additionally, cellulases are known to be useful as a feed additive (see e.g., PCT Publication No. WO 91/04673) and in grain wet milling.
Of primary importance, however, cellulases are used in the treatment of textiles, i.e., in detergent compositions for assisting in the removal of dirt or grayish cast (see e.g., Great Britain Application Nos. 2,075,028, 2,095,275 and 2,094,826 which illustrate improved cleaning performance when detergents incorporate cellulase) or in the treatment of textiles prior to sale to improve the feel and appearance of the textile. Thus, Great Britain Application No. 1,358,599 illustrates the use of cellulase in detergents to reduce the harshness of cotton containing fabrics and cellulases are used in the treatment of textiles to recondition used fabrics by making their colors more vibrant (see e.g., The Shizuoka Prefectural Hammamatsu Textile Industrial Research Institute Report, Vol. 24, pp. 54-61 (1986)). For example, repeated washing of cotton containing fabrics results in a grayish cast to the fabric which is believed to be due to disrupted and disordered fibrils, sometimes called “pills”, caused by mechanical action. This greyish cast is particularly noticeable on colored fabrics. As a consequence, the ability of cellulase to remove the disordered top layer of the fiber and thus improve the overall appearance of the fabric has been of value.
Thus, cellulases have been shown to be effective in many industrial processes. Accordingly, there has been a trend in the field to search for specific cellulase compositions or components which have particularly effective performance profiles with respect to one or more specific applications. In this light, cellulases produced (expressed) in fungi and bacteria have been subject of attention. For example, cellulase produced by certain fungi such as Trichoderma spp. (especially Trichoderma longibrachiatum) have been given much attention because a complete cellulase system capable of degrading crystalline forms of cellulose is readily produced in large quantities via fermentation procedures. This specific cellulase complex has been extensively analyzed to determine the nature of its specific components and the ability of those components to perform in industrial processes. For example, Wood et al., “Methods in Enzymology”, 160, 25, pages 234 et seq. (1988), disclose that complete fungal cellulase systems comprise several different enzyme classifications including those identified as exo-cellobiohydrolases (EC 3.2.1.91) (“CBH”), endoglucanases (EC 3.2.1.4) (“EG”), and β-glucosidases (EC 3.2.1.21) (“BG”). The fungal cellulase classifications of CBH, EG and BG can be further expanded to include multiple components within each classification. U.S. Pat. No. 5,475,101 (Ward et al.) discloses the purification and molecular cloning of one particularly useful enzyme called EGIII which is derived from Trichoderma longibrachiatum. 
PCT Publication No. WO 94/14953 discloses endoglucanases which are encoded by a nucleic acid which comprises any one of a series of DNA sequences, each having 20 nucleotides.
Ooi et al., Curr. Genet., Vol. 18, pp. 217-222 (1990) disclose the cDNA sequence coding for endoglucanase F1-CMC produced by Aspergillus aculeatus which contains the amino acid strings NNLWG (SEQ ID NO: 1), ELMIW (SEQ ID NO: 66) and GTEPFT (SEQ ID NO: 3). Sakamoto et al., Curr. Genet., Vol. 27, pp. 435-439 (1995) discloses the cCNA sequence encoding the endoglucanase CMCase-1 From Aspergillus kawachii IFO 4308 which contains the amino acid strings ELMIW (SEQ ID NO: 66) and GTEPFT (SEQ ID NO: 3). Ward et al., discloses the sequence of EGIII having the amino acid strings NNLWG (SEQ ID NO: 1), ELMIW (SEQ ID NO: 66) and GTEPFT (SEQ ID NO: 3). Additionally, two cellulase sequences, one from Erwinia carotovara and Rhodothermus marinus are disclosed in Saarilahti et al., Gene, Vol. 90, pp. 9-14 (1990) and Hreggvidsson et al., Appl. Environ. Microb., Vol. 62, No. 8, pp. 3047-3049 (1996) which contain the amino acid string ELMIW (SEQ ID NO: 66). However, none of these references discloses or suggests that these amino acid strings have any particular relevance in identifying or isolating other cellulases, and particularly fail to suggest that such cellulases are obtainable from such diverse organisms as bacteria, Actinomycetes and other filamentous fungi.
Despite knowledge in the art related to many cellulase compositions having applications in some or all of the above areas, there is a continued need for new cellulase compositions having improved characteristics which are useful in, for example, treating textiles, as a component of detergent compositions, in the treatment of pulp and paper, food processing, and in the conversion of biomass. Thus, while there has been significant improvement in terms of the understanding of cellulase compositions and their activities, there remains a need for alternative cellulase compositions which retain the beneficial effects of known cellulase compositions. In response to this need, Applicants herein surprisingly discovered that novel microbial enzymes which are related to an enzyme known to be useful in industrial applications, i.e., EGIII, can be detected and obtained by virtue of the presence of unique conserved sequences therein.